A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities Engineered microbial consortia often exhibit enhanced system performance and robustness compared to single-strain biomanufacturing platforms. However, few tools are available for generating co-cultures of the model and key industrial host Saccharomyces cerevisiae. This study engineers auxotrophic and overexpression yeast strains that can be used to create co-cultures through exchange of essential metabolites. Using these strains as modules, two- and three-member consortia were engineered using different cross-feeding architectures. Through ensemble modelling and experimentation, the study explored how cellular and environmental factors govern population dynamics in these systems. The toolkit was tested in a division of labour biomanufacturing case study, showing enhanced and tunable antioxidant resveratrol production. Microbial communities have wide applications in industrial processes and human health. The composition and stability of these systems are influenced by various factors, including environmental characteristics and microbial interactions. Despite their importance, little is known about how communities are established and maintained, which limits our ability to engineer them for human health or industrial purposes. This study presents a toolkit for engineering synthetic S. cerevisiae communities, including 15 auxotrophic strains and 15 target genes for metabolite overproduction. The toolkit enables the creation of novel cross-feeding co-cultures and has wide applications for studying microbial communities and improving bioproduction of high-value compounds. The study identified key engineering targets in co-culture dynamics, including metabolite exchange parameters and initial population ratios. A global sensitivity analysis revealed that these parameters significantly influence co-culture dynamics. The toolkit was used to create synthetic co-cultures with different cross-feeding architectures, including two- and three-member systems. The study demonstrated how different strategies, including metabolite production rates, supplementation, initial population ratios, and cell densities, can control co-culture dynamics. The toolkit was applied to increase resveratrol production by dividing its metabolic pathways between two strains. The study also tested different approaches for controlling population growth rates, final population size, and composition of co-cultures. These approaches included promoter engineering, different initial population ratios, metabolite supplements, and initial cell densities. The results showed that each approach was effective in controlling the growth and population size of synthetic co-cultures. The toolkit was successfully applied to create a metabolic division of labour system for producing a high-value aromatic compound. The study concludes that engineered cross-feeding can be used to construct modular yeast co-cultures, with the toolkit consisting of 15 auxotrophic strains and 15 target genes for metabolite overproduction. The toolkit enables the creation of synthetic co-cultures with distinct features, which can be selected based on the desired application.A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities Engineered microbial consortia often exhibit enhanced system performance and robustness compared to single-strain biomanufacturing platforms. However, few tools are available for generating co-cultures of the model and key industrial host Saccharomyces cerevisiae. This study engineers auxotrophic and overexpression yeast strains that can be used to create co-cultures through exchange of essential metabolites. Using these strains as modules, two- and three-member consortia were engineered using different cross-feeding architectures. Through ensemble modelling and experimentation, the study explored how cellular and environmental factors govern population dynamics in these systems. The toolkit was tested in a division of labour biomanufacturing case study, showing enhanced and tunable antioxidant resveratrol production. Microbial communities have wide applications in industrial processes and human health. The composition and stability of these systems are influenced by various factors, including environmental characteristics and microbial interactions. Despite their importance, little is known about how communities are established and maintained, which limits our ability to engineer them for human health or industrial purposes. This study presents a toolkit for engineering synthetic S. cerevisiae communities, including 15 auxotrophic strains and 15 target genes for metabolite overproduction. The toolkit enables the creation of novel cross-feeding co-cultures and has wide applications for studying microbial communities and improving bioproduction of high-value compounds. The study identified key engineering targets in co-culture dynamics, including metabolite exchange parameters and initial population ratios. A global sensitivity analysis revealed that these parameters significantly influence co-culture dynamics. The toolkit was used to create synthetic co-cultures with different cross-feeding architectures, including two- and three-member systems. The study demonstrated how different strategies, including metabolite production rates, supplementation, initial population ratios, and cell densities, can control co-culture dynamics. The toolkit was applied to increase resveratrol production by dividing its metabolic pathways between two strains. The study also tested different approaches for controlling population growth rates, final population size, and composition of co-cultures. These approaches included promoter engineering, different initial population ratios, metabolite supplements, and initial cell densities. The results showed that each approach was effective in controlling the growth and population size of synthetic co-cultures. The toolkit was successfully applied to create a metabolic division of labour system for producing a high-value aromatic compound. The study concludes that engineered cross-feeding can be used to construct modular yeast co-cultures, with the toolkit consisting of 15 auxotrophic strains and 15 target genes for metabolite overproduction. The toolkit enables the creation of synthetic co-cultures with distinct features, which can be selected based on the desired application.